Patent application title: Lactobacillus Rhamnosus with Body-Fat Reducing Activity and the Foods Containing Them
Yeon-Hee Lee (Seoul, KR)
Yeon-Hee Lee (Seoul, KR)
Kyung-Soo Paek (Seoul, KR)
Kenny Sohn (Gyeonggi-Do, KR)
Tae Jin Kim (Seoul, KR)
Tae Jin Kim (Seoul, KR)
Jee-Hoon Koh (Seoul, KR)
Bum-Suk Park (Seoul, KR)
IPC8 Class: AA61K4500FI
Class name: Whole live micro-organism, cell, or virus containing bacteria or actinomycetales lactobacillus or pediococcus or leuconostoc
Publication date: 2008-10-30
Patent application number: 20080267932
The present invention relates to Lactobacillus strain with a body-fat
reducing activity and provides Lactobacillus rhamnosus Strain PL60
KCCM-10654P. The strain of the invention can be directly used as body-fat
reducing functional foods, or can be used as additives of body-fat
reducing functional foods or a ferment starter strain of body-fat
reducing functional fermented foods. Body-fat inhibiting materials that
the strain of the present invention produce can be isolated to be used.
In addition, in case that fermented foods are produced using the strain
the invention provides conditions capable of having a maximal body-fat
1. A Lactobacillus rhamnosus strain converting linoleic acid into
conjugated linoleic acid.
2. The Lactobacillus rhamnosus strain as set forth in claim 1, wherein said strain is Lactobacillus rhamnosus Strain PL60 KCCM-10654P.
3. The Lactobacillus rhamnosus strain as set forth in claim 1, wherein said strain is a live strain or dried strain.
4. A composition for producing CLA comprising the strain according to claim 1.
5. A mass-producing process of CLA using Lactobacillus rhamnosus strain primary-culturing the strain according to claim 1.
6. The mass-producing process of CLA using Lactobacillus rhamnosus strain as set forth in claim 5, wherein 0.01-1.0% LA or safflower seed oil is added to a primary-culture medium of strain.
7. The mass-producing process of CLA using Lactobacillus rhamnosus strain as set forth in claim 6, wherein 0.01-1.0% Tween-80 is added to a primary-culture medium of strain.
8. The mass-producing process of CLA using Lactobacillus rhamnosus strain as set forth in claim 7, wherein fructose, sucrose, or lactose as a carbohydrate substrate is added to a primary-culture medium of strain.
9. Body-fat reducing functional foods comprising as an additive the strain according to claim 1.
10. The body-fat reducing functional foods as set forth in claim 9, wherein foods are health care foods or fermented foods containing yogurt, cheese, kimchi, kochujang (Korean thick soy paste mixed with red pepper), and doenjang (Korean fermented soy paste).
11. Dairy products prepared using Lactobacillus rhamnosus Strain PL60 KCCM-10654P as a starter strain.
12. Fermented foods from cereals prepared using Lactobacillus rhamnosus Strain PL60 KCCM-10654P as a ferment starter strain.
13. A medicament for preventing and treating obesity-related diseases comprising live strains, dried strains, or cultural filtrates of Lactobacillus rhamnosus Strain PL60 KCCM-10654P.
14. The medicament for preventing and treating obesity-related diseases as set forth in claim 13, wherein healthy people with an average weight of 60 kg take Lactobacillus rhamnosus Strain PL60 KCCM-10654P in an amount of 1.times.10.sup.7-1.times.10.sup.11 CFU per a dose 1-2 times a day.
15. The Lactobacillus rhamnosus strain as set forth in claim 2, wherein said strain is a live strain or dried strain.
16. A composition for producing CLA comprising the strain according to claim 2.
17. A composition for producing CLA comprising the strain according to claim 3.
18. A mass-producing process of CLA using Lactobacillus rhamnosus strain primary-culturing the strain according to claim 2.
19. A mass-producing process of CLA using Lactobacillus rhamnosus strain primary-culturing the strain according to claim 3.
20. Body-fat reducing functional foods comprising as an additive the strain according to claim 2.
The present invention relates to Lactobacillus rhamnosus with body-fat reducing activity and foods containing them. The present invention provides Lactobacillus strains with body-fat reducing activity. The present invention also provides live organisms, killed organisms, broken cell wall fractions, a culture solution, a dried culture solution, a cultured extract containing CLA with a body-fat reducing effect, which result from the Lactobacillus strains of the present invention, and body-fat reducing functional foods and food additives containing them. In addition, the present invention provides body-fat reducing functional foods and beverages using Lactobacillus strain with a body-fat reducing effect as a starter strain or additive. Furthermore, the present invention provides a medicament with a body-fat reducing effect containing the Lactobacillus strains of the present invention.
In addition, the present invention provides conditions capable of maximizing a body-fat reducing effect when fermented foods are produced using the strains of the present invention.
In modern societies, obesity is a disease with lower perfect cure proportion than cancer and increases a death rate as well as various adult diseases resulting from it. It has brought about severe problems enough to make public "war on obesity" in U.S.A. Many materials have been asserted to be a material effective in preventing and treating obesity, but till now only pyruvic acid and conjugated linoleic acid (CLA) have been proved to be efficacious according to a scientific basis (Lenz T L, Hamilton W R. Supplemental products used for weight loss. 2004. J Am Pharm Assoc (Wash DC) 44:59-67). It is suggested that a body-fat reducing mechanism is a reduction of adipose-cell number, a reduction of adipose-cell size, an ingestion reduction of energy and food, a production reduction of fat, an increase of energy consumption, lipolytic activity, an increase of lipid oxidation or like by inducing apoptosis of adipose cells (Chardigny J M, Hasselwander O, Genty M, Kraemer K, Ptock A, Sebedio J L. 2003, Effect of conjugated FA on feed intake, body composition, and liver FA in mice. Lipids. 38(9):895-902).
CLA (c9t11-octadecadienoic acid, t10c12-octadecadienoic acid) is formed through an isomerization of linoleic acid (LA, C18:2 cis9cis12). It has been known that CLA has an antioxidative effect, a cholesterol lowering effect, a growth promoting effect, and an anticancer-effect according to the location of double bonds. Recently, it has been known that CLA has body plasma lipids, a body-fat reducing effect, or like. It has been reported that CLA may be found in animal meats, fermented milk or like. Animal experiments and clinical trials have already proved that especially c9, t11-CLA of CLA isomers has a body-fat reducing effect. Most ideally, c9t11-CLA and t10c12-CLA are most preferably produced in the same quantity.
Butyrivibrio fibrosolvents is the first found anaerobic microorganism that produces CLA, which is isolated from ruminants like a cow. It produces trans-11-Octadecenoic acid through 2 steps upon the biohydrogenation of LA. cis-9, trans-11-Octadecadienoic acid is produced by the action of linoleic acid isomerases, prior to hydrogenating the generated conjugated acid to produce trans-11-octadecenoic acid.
According to the recent Norway study in 2004 (Gaullier J M, Halse J, Hoye K, Kristiansen K, Fagertun H, Vik H, Gudmundsen O. 2004. Conjugated linoleic acid supplementation for 1 y reduces body fat mass in healthy overweight humans. Am J Clin Nutr. 79(6):1118-1125), CLA caused a weight loss of 4-10% without side effects when administered to 180 overweight people for one year.
The present invention selected and identified a Korean-type Lactobacillus strain with a body-fat reducing effect that overproduced t10c12-CLA, confirmed characteristics of a probiotic, such as intestinal adaptation or like, in the strain, and found out conditions that the strain could maximally produce CLA and Lactobacillus strains with a body fat reducing effect by carrying out an animal experiment to confirm weight loss.
Therefore, the present invention has been made in view of the above problems, and it is an object of the present invention to provide a strain that produces CLA.
The strain of the present invention is Lactobacillus rhamnosus Strain PL60 that was deposited as KCCM-10654P to Korean Culture Center of Microorganisms on May 9 h, 2005.
Another object of the present invention is to provide Lactobacillus strains capable of reducing body fat.
Still another object of the present invention is to prevent or treat various adult diseases by reducing body fat.
Further another object of the present invention is to provide conditions that produce maximum CLA with a body-fat reducing effect.
Additional another object of the present invention is to provide a strain that has a body-fat reducing effect, good adhesion to the intestines, and strong tolerance to both acid and bile.
Further still another object of the present invention is to provide as a probiotic Lactobacillus strains that doesn't transfer an antibiotic resistance and is harmless.
Lactobacillus strains can be prepared in various compositions, preferably these compositions are compositional forms, such as capsules, tablets, powder etc and convenient forms capable of being added to various foods.
These formulations can be prepared using pharmaceutically acceptable carriers, excipients, solvent or supplements by the known methods. These method and ingredients have been well known, and are in detail disclosed in standard texts and manuals, for example a publication (Remington. 1995. The Science and Practice of Pharmacy. Mack Publishing Co. Easton, Pa. 18042, USA), which is incorporated herein by reference.
Digestive Foods containing Lactobacillus strains may be prepared by the well-known method in the art.
Foods and beverages with a body-fat reducing effect may be prepared by the well-known method in the art using the strain as a starter strain or additive of fermented foods containing fermented milk products.
Fermented foods with a maximum body-fat reducing effect can be produced using conditions suggested herein.
Hereinafter, the present invention is explained in more detail.
In accordance with an aspect of the present invention, the above and other objects can be accomplished by the provision of body-fat reducing functional foods.
In accordance with another aspect of the present invention, there is provided Lactobacillus rhamnosus Strain PL60 KCCM-10654P capable of reducing body fat.
In accordance with another aspect of the present invention, there are provided body-fat reducing functional foods containing Lactobacillus rhamnosus Strain PL60 KCCM-10654P in an amount of 1×106-1×1011 CFU/g in order to prevent and treat adult diseases using a body-fat reducing effect.
In accordance with another aspect of the present invention, there are provided food and beverage additives containing Lactobacillus rhamnosus Strain PL60 KCCM-10654P.
In accordance with another aspect of the present invention, there are provided conditions capable of obtaining a maximum body-fat reducing effect in fermented foods using Lactobacillus rhamnosus Strain PL60 KCCM-10654P.
Hereinafter, the present invention will be described in more detail by reference to examples of preferred embodiments of the present invention which, however, are not to be construed as limiting the present invention in any way.
Lactobacillus rhamnosus Strain PL60 of the present invention has a body-fat reducing effect to be capable of preventing or treating diseases resulting from obesity. In addition, dried Lactobacillus rhamnosus Strain PL60 and Lactobacillus rhamnosus Strain PL60 cultural filtrates, dried cultural filtrates of the present invention may be used as additives of various foods and beverages to be useful in preventing and treating body fat, hence can be used in preventing and treating all obesity-related diseases. Furthermore, fermented foods using said Lactobacillus rhamnosus Strain PL60 of the present invention could prevent and treat obesity by a body-fat reducing effect.
In addition, according to the present invention Lactobacillus rhamnosus Strain PL60 must be primary-cultured in a medium containing LA in order to produce maximum CLA. LA content is 100-1000 ppm, Tween-80 content is 1-0.1%, and carbohydrate is preferably fructose, sucrose, and lactose, most preferably fructose, so that fermented foods using Lactobacillus rhamnosus Strain PL60 have a maximum body fat reducing effect.
DESCRIPTION OF THE DRAWINGS
The above and other objects, features and other advantages of the present invention will be more clearly understood from the following detailed description taken in conjunction with the accompanying drawings, in which:
FIG. 1 is a gas chromatogram identifying CLA generated by Lactobacillus rhamnosus Strain PL60.
FIG. 2 is a micrograph of Lactobacillus rhamnosus Strain PL60.
FIG. 3 shows the 16S rRNA sequence of Lactobacillus rhamnosus Strain PL60.
FIG. 4 is band patterns identifying Lactobacillus rhamnosus Strain PL60 using a multiplex PCR.
FIG. 5 shows the experimental results for an adaptation of Lactobacillus rhamnosus Strain PL60 to Caco-2 cells.
FIGS. 6a and 6b show the experimental results for an adhesion of Lactobacillus rhamnosus Strain PL60 to the human intestines.
FIG. 7 is band patterns illustrating PCR results of an isolated colony after orally administrating Lactobacillus rhamnosus Strain PL60 to people.
FIG. 8 shows the changes of the body weight of rats that took Lactobacillus rhamnosus Strain PL60.
FIG. 9 is a graph comparing the body weight of rats of each group after administrating Lactobacillus rhamnosus Strain PL60 on the 10th week.
Screening of Lactobacillus Strains Capable of Producing Conjugated Linoleic Acid (Hereafter, Referred to CLA)
In order to select CLA-producing strains, Lactobacillus strains that grew in a medium containing LA, a substrate of CLA, were screened. And then, it was confirmed whether they expressed an isomerase enzyme, an enzyme involved in producing CLA.
Materials and Method>
First, Lactobacillus strains that grew in a medium containing linoleic acid (LA) were selected, of which CLA-producing Lactobacillus strains were screened. For this, CLA-producing strains may easily be screened from a large quantity of Lactobacillus strains by using an isomerase assay (Ogawa J, Matsumura K, Kishino S, Omura Y, and Shimizu S. 2001. Conjugated linoleic acid accumulation via 10-Hydroxy-12-octadecaenoic acid during microaerobic transformation of linoleic acid by Lactobacillus acidophilus. Appl. Envir. Microbiol. 67:1246-1252). First, Lactobacillus strains that grew in a MRS medium containing 0.1% LA were primarily selected. And then, these Lactobacillus strains were twice subcultured in a MRS broth and cultured in a MRS broth containing 0.1% LA 10 mL for 2 days. The medium of 5 mL was centrifuged at 800 rpm for 10 minutes to collect cells, prior to washing the cells with a 0.1M potassium phosphate buffer solution (pH 7.0). Again, thereto a 0.1M potassium phosphate buffer solution (pH 7.0) 11.0 mL was added, followed by breaking and centrifuging the admixture every 3 minutes in a cold state using an ultrasonic breaker to obtain a crude enzyme solution. The crude enzyme solution was added to a substrate solution (LA 0.1 mL, 0.1M potassium phosphate buffer 2.7 mL, and 1,3-propanediol 0.2 mL) to measure an absorbance at 233 nm.
Results and Discussion>
CLA-producing Lactobacillus strains were screened out of more than 200 Lactobacillus strains using an isomerase assay.
Experimental Example 1
Identification of CLA Production Using a Gas Chromatography
In order to confirm how much CLA was substantially produced by Lactobacillus strains expressing isomerase enzymes, the quantity of generated CLA was determined using a gas chromatography.
Materials and Method>
Lactobacillus candidates were inoculated into a MRS liquid medium containing LA, prior to culturing the mixture at 37° C. for 24-48 hours. The cultured medium was extracted with heptadecanoic acid and a mixture of chloroform:methanol. The extract was treated with sodium sulfate to remove moisture, and then evaporated. 1N Sodium hydroxide (in methanol) was added to the prepared sample, prior to saponifying the sample at 100° C. for 15 minutes. Thereto 4% HCl (in methanol) was added to be methylated. Hexane:water (1:1, v/v) were added to the methylated sample, and then mixed and centrifuged. An organic solvent fraction was taken to remove organic solvent using nitrogen gas, followed by dissolving the sample in hexane 1 mL.
According to the present invention, CLA content within each sample before and after the removal of oxides was measured by gas chromatography (Hewlett Packard 5890 Series II GC) with a flame ionization detector (FID). The used capillary column (DB FFAP capillary column) has a length of 30 m, an inner diameter of 0.25 μm, and a film thickness of 0.25 μm. After setting the column into a GC, a GC was used under the following conditions: oven temperature (210° C.); detector temperature (270° C.); injector temperature (250° C.); carrier gas (Helium (1 mL/min)); split ratio (50:1); and sample injection (2 μl). Each peak area was calculated using an integrator (3395, Hewlett Packard) linked with the GC. CLA was identified, as compared with the retention time of a standard material. Heptadecanoic acid was used as an internal standard material in order to measure CLA contents (Lin, T. Y. 2000. Conjugated linoleic acid concentration as affected by lactic cultures and additives, Food Chemistry 69. 27-31).
Results and Discussion>
As indicated in the gas chromatogram of FIG. 1, the isolated Lactobacillus strain produced both c9t11 and t10c12 isomers of CLA. If yield of t10c12 CLA with a body-fat reducing effect was indicated in terms of ppm, Lactobacillus rhamnosus Strain PL60 produced t10c12 CLA in an amount of 43.25 ppm and had more excellent productivity in comparison with the reported Lactobacillus reuteri and Lactobacillus fermentum that produced the CLA in amounts of 30 ppm and 28 ppm respectively.
Experimental Example 2
Identification of Lactobacillus strain: Gram's Staining, Identification Using API Kit, 16S rRNA Sequence Analysis, Multiplex PCR Assays
In order to identify CLA-producing Lactobacillus strains, it was confirmed whether they showed gram positive on a gram's staining and catalase negative or not. Various biochemical and physiological tests were carried out using an API kit, and 16S rRNA sequence was analyzed and identified. In addition, in order to classify closely related species, strains were identified by multiplex PCR assays using a group-specific primer.
1. Gram's Staining
Straining was smeared on a slide and heat-fixed, prior to adding a crystal violet solution thereon to be reacted for about 1 minute. The resulting slide was treated with an iodine solution to wash an excess of dyes, followed by adding iodine thereon to be treated for 1 minute. The resulting slide was decolorized with 95% ethanol for 30 seconds, and then washed with water for 2-3 seconds to remove water with a sucker. The resulting slide was treated with a safranin 0 solution for 10-30 seconds for a counter stain. The resulting slide was carefully rinsed with water until dyes didn't come out any more, followed by drying the resulting slide with a sucker and letting a drop of immersion oils fall to be observed through a microscope.
Results and Discussion>
As shown in FIG. 2, CLA-producing Lactobacillus stains exhibited gram positive.
2. Biochemical and Physiological Tests Using an API Kit.
After confirming whether strains were purely isolated or not, the strains were cultured in a MRS medium at 30° C. or 37° C. for 24 hours. They were more than twice subcultured in a MRS broth, prior to isolating a colony from a MRS medium. A suspension medium ample was opened to prepare a heavy suspension with very high turbidity using a cotton ball. The prepared strain solution was dropped into the suspension medium 5 mL drop by drop till turbidity reached McFarland 2. The API 50 CHL medium containing the strains was divided into tubes of a strip and cultured under the aerobic condition at 30° C. or 37° C. for 48 hours. If acid is generated, an API kit makes a medium yellowish by a bromocresol purple indicator within the medium. If color changes from purple to black in an Esculin test (Tube No. 25), it means a positive reaction.
Results and Discussion>
As indicated Table 1, experimental results using an API 50CH kit showed that the Lactobacillus strain of the invention had a similarity to Lactobacillus rhamnosus and Lactobacillus para paracasei, but it wasn't evaluated in terms of %.
TABLE-US-00001 TABLE 1 Results on identification of Lactobacillus strain using API CH50 kit Strip No. 1 Strip No. 1 Strip No. 1 Strip No. 1 Strip No. 1 Tube/substrate Tube/substrate Tube/substrate Tube/substrate Tube/substrate -Control +Galactose -D-Mannoside -Melibiose -D-Turanose -Glycerol +D-Glucose -D-Glucoside -Saccharose -D-Lyxose -Erythritol +D-Fructose +Glucosamine +Trehalose +D-Tagatose +D-Arabinose +D-Mannose +Amygdaline -Inuline -D-Fucose -L-Arabinose -L-Sorbose +Arbutine +Melizitose +L-Fucose +Ribose -Rhamnose +Esculine -D-Raffinose -D-Arabitol -D-Xylose -Dulcitol +Salicine -Amidon -L-Arabitol -L-Xylose -Inositol +Cellobiose -Glycogene +Gluconate -Adonitol +Mannitol -Maltose -Xylitol -2-Gluconate -Xyloside +Sorbitol -Lactose +Gentiobiose -5-Gluconate Lacto. para. Paracasei 3 Lacto. papa. Paracasei 1
3. Identification Using 16S rRNA Sequence Analysis
Genomic DNA was isolated to amplify a 16S ribosomal DNA fragments thereof, prior to confirming the amplified DNA fragments by an electrophoresis. DNA fragments were purified using a Qiagen PCR purification kit (Qiagen, Hilden, Germany) to be mixed with a reactant solution containing d-Rhodamine dye-labeling dd-NTP, prior to performing a direct sequencing to purify the obtained DNA using an ethanol/sodium acetate precipitation. The purified DNA was dissolved in TSR (template suppression reagent) to be analyzed with an ABI prism 310 Genetic analyzer (PE Applied Biosystems, U.S.A). The analyzed sequence was identified using Genebank (http://www.ncbi.nlm.nih.gov/).
Results and Discussion>
As a result of analyzing the sequence of CLA-producing Lactobacillus strain (FIG. 3), it showed a similarity to Lactobacillus rhamnosus 842/844 (99%) and Lactobacillus casei (99%).
4. Identification Using Multiplex PCR
In order to confirm whether the PL60 strain was Lactobacillus rhamnosus or Lactobacillus casei(Lactobacillus paracasei), the DNA fragments obtained from the PL60 strain were compared with their DNA fragments after performing multiplex PCR assays (Song, Y., N. Kato, C. Liu, Y. Matsumiya, H. Kato, and K.
Watanabe. 2000. Rapid identification of 11 human intestinal Lactobacillus species by multiplex PCR assays using group- and species-specific primers derived from the 16S, 23S rRNA intergenic spacer region and its flanking 23S rRNA. FEMS Mictrobiol. Letters, 187:167-173). For this, a PCR reaction was carried out in a final amount of 300 using a mixture containing 1× reaction buffer, dNTPs of 200 μM, Taq polymerase of 0.15 units, primers of 10 pmol (LU-5, CTA GCG GGT GCG ACT TTG; Lpar-4, GGC CAG CTA TGT ATT CAC TGA; Rha II, GCG ATG CGA ATT TCT ATT ATT), and DNA of 20 ng. The PCR reaction comprised the following steps of: reacting the mixture repeating a cycle consisting of 20 seconds at 95° C., 2 minutes at 62° C., and 2 minutes at 74° C. 35 times; reacting the mixture at 72° C. for 10 minutes; and preserving the resultant at 4° C. The PCR resultant was electrophoresed in 1.5% agarose gel for 20 minutes using a 0.5×TBE (0.045M tris-borate, 0.001M EDTA) buffer solution, followed by observing the patterns of the developed DNA fragments.
Results and Discussion>
As a result of multiplex PCR assays, Lactobacillus rhamnosus strain and the PL60 strain produced DNA fragments of 113bp, whereas Lactobacillus paracasei ATCC 25302 produced DNA fragments with a size of 312 bp as shown in FIG. 4. Consequently, the PL60 strain was identified as Lactobacillus rhamnosus. Up to now, CLA-producing Lactobacillus rhamnosus strain hasn't been reported yet. The present invention reports CLA-producing Lactobacillus rhamnosus strain for the first time.
Experimental Example 3
Intestinal Adaptation of Lactobacillus rhamnosus
In order to be used as a probiotic, it must have strong tolerance to both acid and bile and good adaptation to intestinal cells. An intestinal adaptation should be confirmed through human experiments.
1. Acid Resistance Test
In order to know if pH affected survivability of selected strains, a MRS (DeMan-Rogosa-Sharpe) medium was used after adjusting pH to 7.0, 4.8, and 4.5 using 10N HCl. An activated strain solution (0.D=2.0) was inoculated into a MRS medium in an amount of 2% and cultured at 37° C. for 24 hours, prior to measuring an absorbance at 600 nm. It was examined if pH affected growth of selected strains using the measured absorbance. The 0.D of pH 7.0 was diluted to 1/10 to measure and record an absorbance (Conway P L, Gorback S L, Goldin B R, 1987. Survival of lactic acid bacteria in the human stomach and adhesion to intestinal cells. J. Dairy Sci. 70:1-12).
Results and Discussion>
As a result of an experiment on survivability in the presence of low acid, even if said strains were treated for 24 hours, they survived, hence had a strong resistance to acid as shown in Table 2.
TABLE-US-00002 TABLE 2 Experimental results on acid resistance of Lactobacillus rhamnosus Strain PL60(0.D. at 600 nm) Time 0 hr 3 hr 6 hr 24 hr PH 7.0 0.022 0.069 0.467 8.080 PH 4.8 0.036 0.063 0.249 6.850 PH 4.5 0.029 0.064 0.200 5.820
2. Bile Resistance Test
In order to know if bile affected growth of selected strains, ox-gall (OXOID) was added to a MRS (DeMan-Rogosa-Sharpe) medium in amounts of 0.125% and 0.25% to be sterilized. The activated strain solution (0.D=2.0) was inoculated into the sterilized medium in an amount of 2% and cultured at 37° C. for 24 hours, followed by measuring an absorbance at 600 nm. The 0.D in 0% bile was diluted to 1/10 to measure and record an absorbance (Ibrahim S A, Bezkorovainy A. 1993. Survival of bifidobacteria in the presence of bile salt. J. Sci. Food Agric. 62: 351-354).
Results and Discussion>
Healthy people have a bile concentration of 0.06% within the small intestines. The strains survived even in the presence of 0.250% bile, thus had a strong bile resistance.
TABLE-US-00003 TABLE 3 Time 0 hr 3 hr 6 hr 24 hr Bile 0.000% 0.022 0.069 0.467 8.080 Bile 0.250% 0.007 0.038 0.335 3.570
3. Intestinal Adhesion Test
In order to know an adhesion to the human intestines, Lactobacillus rhamnosus Strain PL60 was adhered to Caco-2 cell lines derived from intestinal epidermal cells. For this, Caco-2 cell lines were cultured in a DMEM medium (pH 7.0) containing sodium bicarbonate 2.7 g/L, 20% (v/v) fetal bovine serum (FBS) and antibiotics antimicotics. 3×105 Cells were inoculated into a medium of 2 mL in a petri dish of 30 mm to be cultured into a single layer. The medium was changed once every two days. The cell single layer was twice rinsed with a phosphate buffered saline (PBS) solution of 2 mL, 6 days later. The Lactobacillus strain of 1×107 cells was suspended in a medium of 2 mL and added to a petri dish, prior to culturing the admixture at 37° C. under an 5% CO2-95% air atmosphere for 60-90 minutes. The cells were twice rinsed with a sterilized PBS and fixed with methanol for 10 minutes. They were observed through an optical microscope after a gram's stain. 20 Fields were inspected under a 100-fold microscope for a quantitative analysis. The number of adhered strains was counted and indicated in terms of the number of adhered strains per 100 Caco-2 cells (Bibiloni R, Perez P F, DeAntoni G L. 1999. Anaerobe 5, 483-485; Edited by R. Fuller (1997) Probiotics 2, 10-22).
Results and Discussion>
As shown in FIG. 5, Lactobacillus rhamnosus Strain PL60 has excellent adhesion to the Caco-2 cells. If the number of adhered strains per a field was counted out of 20 fields to calculate an average number of adhered strains per a field, 59.29±5.33 Lactobacillus strains per a field were adhered. This means that more than 4000 of Lactobacillus strains per a petri dish were adhered to the cells and had better intestinal adhesion than the conventional Lactobacillus strains.
4. Adaptation Test to the Human Intestines
In order to confirm whether Lactobacillus strains were adapted to the intestines after people substantially took them, Lactobacillus rhamnosus Strain PL60 was orally administered in an amount of 1010 CFU once a day for 8 days. The next day, feces were cultured in a MRS (with 1% bromo phenol blue, 30 μg/mL vancomycin) for 48 hours. All the similar colonies were examined by a gram's stain, subcultured, and purely isolated. Species-specific PCR assays were carried out using purely isolated colonies.
Results and Discussion>
As shown in FIG. 6, Lactobacillus rhamnosus Strain PL60 had been detected from one day to the six days after taking it and stopped an administration as soon as it was detected. The detected Lactobacillus colony turned out to be Lactobacillus rhamnosus by a species-specific PCR assay (FIG. 7). This proves that Lactobacillus rhamnosus Strain PL60 was adapted to the intestines. Especially, as shown in FIG. 6, it was thought that judging by the fact that bacterial florae within the intestines got simpler after Lactobacillus rhamnosus Strain PL60 was administered, the Lactobacillus strain had an intestinal regulation.
Experimental Example 4
Safety Test of Lactobacillus Strains
The safety test of Lactobacillus strains should be carried out for human dosage. For this, it was confirmed whether Lactobacillus strains produced toxic materials, such as ammonia, indole, hemolysin or like or not, and poisonous enzymes were present or not.
1. Hemolysis Test
When Lactobacillus rhamnosus Strain PL60 was inoculated into a sheep blood agar and cultured at 37° C. for 24 hours, only α-hemolysis was found, not β-hemolysis.
2. Gelatin Liquefaction Test
Lactobacillus rhamnosus Strain PL60 was inoculated into a slant medium made of a MRS gelatin medium (beef extract of 0.3 g, peptone of 0.5 g, gelatin of 12 g, and MRS broth of 100 mL) and cultured at 35° C. for 6 weeks. When it, together with a control, was cooled at 4° C. for 4 hours or so to examine gelatin liquefaction, it was thought that gelatinases were not present because a gelatin liquefaction wasn't observed.
3. Ammonia Formation Test
A urea agar medium (urea of 20 g, NaCl of 5 g, KH2PO4 of 2 g, peptone of 1 g, glucose of 1 g, phenol of 12 mg, and distilled water of 100 mL) was filtered and sterilized, followed by dissolving agar of 15 g in distilled water of 900 mL to be wet-sterilized and mixed with the prepared urea agar medium to adjust a total volume to 1 L (pH 6.9). Thereto Lactobacillus rhamnosus Strain PL60 was inoculated and cultured at 37° C. for 12 hours or so, prior to observing color change of the medium. Because a yellow medium means negative, it was proved that Lactobacillus rhamnosus Strain PL60 didn't generate ammonia.
4. Indole Formation Test
Lactobacillus rhamnosus Strain PL60 was inoculated into a MRS agar containing 0.1% tryptone and cultured for 18 hours or so. When thereto 5 drops of a Kovac's reagent (p-dimethylaminobenzaldehyde of 10 g, buthanol of 150 mL, and hydrocholic acid of 50 mL) were added, there was no color change. This means that indole wasn't produced.
5. Phenylalanine Deamination Test
Lactobacillus rhamnosus Strain PL60 was inoculated into a MRS medium containing 0.2% D,L-phenylalanine and cultured for 24 hours or like. After thereto letting 5-10 drops of 10% ferric chloride fall to flow down on a slant medium, a color change was observed within 1-5 minutes. In case of a positive reaction, the generated phenylpyiuvic acid was reacted with ferric chloride to make a medium green. Lactobacillus rhamnosus Strain PL60 showed a negative reaction.
6. β-Glucuronidase Test
p-Nitrophenyl-4-D-glucuronide was dissolved in 0.1M sodium phosphate buffer (pH 6.0) for a 0.2% concentration. Lactobacillus rhamnosus Strain PL60 was suspended in a phosphate buffer to Ab600=4 to form a suspension. A buffer solution of 200 μl with a substrate was added to the suspension of 200 μg and treated at 37° C. for 16 hours. If a culture solution gets yellow, it is positive. However, this test showed a negative reaction. The culture solution was centrifuged to take a supernatant. When an absorbance of the supernatant was determined at 405 nm, it was 0.078.
7. Nitroreductase Activity Test
Lactobacillus rhamnosus Strain PL60 cultured in a MRS liquid medium overnight was centrifuged at 3000×g for 10 minutes to collect biomass, prior to sonicating the biomass for 5 minutes. 4-Nitrobenzoic acid (final conc. 30 μg/mL) and trichloroacetic acid (final conc. 0.21%) were added to the supernatant and treated at 37° C. for 1 hour, followed by adding sodium nitrite (final conc. 0.007%) to be treated at room temperature for 20 minutes. Thereto ammonium sulfamate (final conc. 0.04%) was added and treated at room temperature for 3 minutes. Thereto NEDD (N-(1-naphtyl)ethylenediamine dihydrdchloride) (final conc. 0.35%) was added and developed at 4° C. When the developed supernatant was observed under a 540 nm spectrophotometer, it showed a negative reaction. It was compared with a positive reaction obtained from adding 4-aminobenzoic acid of 1 μg/mL.
8. Antibiotic Resistance
The stronger antibiotic resistance a probiotic has, the higher survivability within the intestines is. Thus, the stronger an antibiotic resistance is, the better it is. However, if an antibiotic resistance is transferred, resistance problems may be brought about. It was confirmed whether an antibiotic resistance was transferred to other bacteria or not.
TABLE-US-00004 TABLE 4 Antibiotic resistance of Lactobacillus rhamnosus Strain PL60 Antibiotic Diameter(mm) of growth inhibition Ampicillin 21 Carbenicillin 25 Cefoperazone 22 Cephalothin 20 Chloramphenicol 24 Clindamycin 26 Erythromycin 28 Gentamicin 10 Oxacillin 8 Penicillin 25 Piperacilin 30 Rifampin 24 Streptomycin 11 Tetracycline 29 Trimethpprime/sulfamethoxasole 6 Vancomycin 6
9. Transfer Test of Antibiotic Resistance
In order to examine the transfer of an antibiotic resistance, a filter binding assay was carried out (Givers, D., G. Huys, and J, Swings. 2003. In vitro conjugal transfer of tetracycline resistance from Lactobacillus isolates to other Gram-positive bacteria. FEMS Microb. Letters 225:125-130). Lactobacillus rhamnosus Strain PL60 was cultured to a mid-exponential phase (approximately 4-5 hours). The cultured strain of 1 mL was mixed with Enterococcus faecalis CCARM 5510 of 1 mL, followed by filtering the mixture through a sterilized cellulose acetate filter to be washed with PPS (peptone physiological saline solution). The filter paper was put on a non-selective agar medium and cultured at 37° C. for 16 hours. Biomass grown on the filter paper was washed with PPS of 2 mL and detached from the paper, prior to diluting the biomass to be inoculated into an Enterococcosal selective medium containing various antibiotics and cultured at 37° C.; for 24-48 hours. It was examined whether E. faecalis with an antibiotic resistance was present or not, but there was no E. faecalis with an antibiotic resistance in the culture. This means that the antibiotic resistance was not transferred.
MODE FOR INVENTION
Optimum Conditions for Producing CLA
We found the concentration of LA and the kind of substrates that can maximally produce CLA.
1. LA Concentration Capable of Producing Maximum CLA
As LA of high concentration inhibits the growth of bacteria themselves, LA can't be added to a medium in high concentration. In addition, in order to save LA spent on a medium LA concentration that could produce maximum CLA was found out.
Materials and Method>
Water-soluble LA ester was added to a skim milk medium and MRS medium for various concentrations and cultured overnight, followed by measuring the quantity of CLA generated within the media. For this, lipid within a medium was extracted and methylated, prior to measuring the quantity of generated CLA using a GC. To do this, heptadecanoic acid of 1000 ppm and chloroform:methanol (2:1) of 200 mL were added to a culture solution of 20 mL, followed by thereto adding glass beads to be strongly shaken for 5 minutes and homogenized for 5 minutes.
The admixture was centrifuged at 6000 rpm for 15 minutes (4° C.) and separated into two fractions. An organic solvent fraction was treated with sodium sulfate to remove residual moisture, prior to evaporating organic solvent to be dried with nitrogen gas. 1N Sodium hydroxide (methanol) of 3 mL was added to the dried sample and saponified at 100° C. for 15 minutes. At this time, a screw-capped tube treated with a Teflon tape was used and the cap was wrapped with a parafilm. Thereto 4% HCl (methanol) of 6 mL was added to be methylated for 20 minutes. The methylated sample was mixed with hexane:water (1:1, v/v) of. 2 mL and strongly shaken for 10 minutes, followed by centrifuging the mixture at 800 rpm and 4° C. for 15 minutes. An organic solvent fraction was taken and dried using nitrogen gas, prior to dissolving the dried matter in hexane 1 mL.
Results and Discussion>
Supposing that the peak area of heptodecanoic acid, a standard reference material, is 100, when LA was in an amount of more than 100 ppm added to a medium CLA was produced in a sufficient amount (Table 5). In addition, if LA was in amounts of 1000 ppm and 500 ppm each added there was no striking difference between them in producing CLA. Preferably, LA is in an amount of 100-1000 ppm added in order to produce CLA. In the view of cost and efficiency, 500 ppm is most preferable.
TABLE-US-00005 TABLE 5 CLA production according to LA concentration added to a medium Retention LA concentration added to medium (ppm) time (min.) 0 ppm 10 ppm 100 ppm 500 ppm 1000 ppm 6.867 Heptadecanoic 100 100 100 100 100 acid 12.002 CLA (c9, t11) 12690 12730 14850 15250 15779 12.332 CLA (t10, c12) 3984 3990 5435 8854 9727 13.000 8211 8158 8148 8639 8573
2. Emulsifier Addition Conditions for Producing Maximum CLA
It was examined if when an emulsifier was added in order to increase the solubility of LA in a culture solution, the production of CLA increased or not. For this, LA was added to a skim milk medium and MRS medium for a 0.1% concentration. At this time, LA was added in three form of LA, LA salt, and LA and Tween-800 (0.2%) and cultured overnight, followed by confirming the CLA productivity of Lactobacillus rhamnosus Strain PL60. Using the above-mentioned method, lipid within a culture solution was extracted to be methylated, prior to determining the quantity of CLA by GC.
Results and Discussion>
A Tween-80 that was used in order to enhance a solubility of LA in a culture solution tripled the production of t10c12 CLA, as compared with a LA salt. It is very important that an emulsifier was added to enhance solubility of LA upon adding LA to a medium.
TABLE-US-00006 TABLE 6 Influence of Tween-80 addition on CLA production Lactobacillus rhamnosus Strain PL60 Skim Skim Skim milk + LA Skim milk + RT(min) milk(blank) milk salt LA + Tween 80 6.859 414941 406823 455181 448288 12.010 8414 13296 19408 25817 12.328 2323 3797 4396 12559 13.000 4505 7463 11330 13728
3. Emulsifier Addition Conditions upon Primary culture for Inducing CLA production
In order to produce maximum CLA immediately after taking Lactobacillus rhamnosus Strain PL60 itself, or a starter strain or additive thereof, it was examined whether in case Lactobacillus rhamnosus Strain. PL60 was cultured to produce products like lyophilized-dry powders, adding Tween-80 to increase solubility of LA was an efficient condition or not. For this, LA salt, LA and Tween-80 of 0.1%, LA and Tween-80 of 0.2%, and LA and Tween-80 of 0.5% were added to a medium on primary-culturing starter strains. The primary-cultured Lactobacillus rhamnosus Strain PL60 was cultured in a CLA-producing medium (skim milk containing LA of 0.1%) to measure the quantity of the generated CLA.
Results and Discussion>
In order to produce maximum CLA in a skim milk medium (whey medium) used in a commercial production, in case Lactobacillus rhamnosus Strain PL60 was cultured in a skim milk medium containing LA of 0.1% and Tween-80 of 0.1-0.5% to induce productivity of CLA, CLA productivity was best (Table 7). It was thought that the reason why 0.2% Tween-80 has higher CLA productivity than 0.5% Tween-80 was the growth inhibition of Lactobacillus strains by 0.5% Tween-80.
TABLE-US-00007 TABLE 7 CLA productivity of Lactobacillus rhamnosus Strain PL60 depending on concentrations of Tween-80 for dissolving LA in a medium Skim milk containing 0.1% LA Control LA + Tween80 LA + Tween80 LA + Tween80 LA + Tween80 (without LA) LA salt (0.01%) (0.1%) (0.2%) (0.5%) 6.859 100 100 100 100 100 100 12.010 5980 6901 12846 15972 18610 14886 c9t11 12.320 2398 3229 4110 4184 6562 4006 t10c12 13.000 4450 5161 10313 8912 10183 8786
4. Saccharide-Addition Conditions for Producing Maximum CLA
We found out the kind of saccharides capable of producing maximum CLA. To do this, fructose, sucrose, and lactose each was added to a skim milk containing 0.1% LA medium for a 6% concentration to measure a production of CLA.
Results and Discussion>
CLA was produced most on adding fructose, followed by sucrose and lactose. When glucose was added, an effective CLA production was not observed.
TABLE-US-00008 TABLE 8 Change of CLA production depending on various saccharides Skim milk RT without PL60 Control lactose fructose glucose sucrose 6.895 30968 459810 496664 447001 381032 468243 12.091 28760 40559 44592 46703 33612 43901 12.419 17850 24097 24201 29864 14860 23519
Change of the Body Weight of Rats Administered with CLA-Producing Lactobacillus rhamnosus Strain PL60
A lyophilized Lactobacillus rhamnosus Strain PL60 that was cultured in a medium containing 0.1% LA and 0.2% Tween-80 using skim milk as an excipient was administered into a rat in a dose of 109 CFU/day and 107 CFU/day with giving a high-fat diet, followed by observing the change of body weight of a rat.
Materials and Method>
Four C57BL/6N rats (Charles river laboratory animal facility, USA) were assigned to five groups. The first group was a group administered with a normal diet (Purina rodent chow #5057 (3.28 cal/g), the second group was a group administered with a high-fat diet (Research diet 45% high fat diet D12451 (5.252 cal/g), the third group Was a control group administered with a high-fat diet and skim milk of an excipient, the fourth group was a group administered with a high-fat diet and Lactobacillus rhamnosus Strain PL60 in high concentration (109 CFU/day), and the fifth group was a group administered with a high-fat diet and Lactobacillus rhamnosus Strain PL60 in low concentration (107 CFU/day). While 3 week-old rats ate a high-fat diet and water to the full, the change of their body weight and the quantity of a fed diet were observed. The rats were anatomized on the 8th week to observe weight of intestinal fat, the size and number of adipose cells in all organs using a microscope after a stain.
Results and Discussion>
Table 9 represents the change of body weight of rats administered with Lactobacillus rhamnosus Strain PL60. According to Table 9, while a group administered with Lactobacillus rhamnosus Strain PL60 in high concentration, hardly showed a significant statistic on the 4th week, it had lower weight gain by more than 2 g on the 8th week, as compared with a control group (FIG. 8 and FIG. 9). That is to say, a normal-diet group had an average weight of 24.7 g, a high fat-diet group had an average weight of 33.4 g, a skim-milk group had an average weight of 31.9 g, a group administered with Lactobacillus rhamnosus Strain PL60 in high concentration had an average weight of 26.9 g, and a group administered with Lactobacillus rhamnosus Strain PL60 in low concentration had an average weight of 28.7 g. The weight gain of the high-concentration group was lower than that of the high fat-diet group by 6.5 g, which was 19.5%. The low-concentration group had a lower weight gain than the high fat-diet group by 4.7 g, which was 14%. The high-concentration group and low-concentration group respectively showed lower weight gain by 5 g (15.7%) and 3.2 g (10%), as compared with a skim milk group. It was thought that the weight difference between a high fat diet group and a skim milk group was 1.5 g (4.5%), which was in the tolerance error range and was not a weight loss resulting from skim milk.
TABLE-US-00009 TABLE 9 6/11 No. 3 weeks 6/17 6/18 6/25 6/28 7/2 7/5 7/9 7/13 7/16 7/20 7/27 7/30 NC 1 9.8 14.8 16 19.9 20 22 22 23 22.3 23 22.1 24.2 23.5 2 9 123. 15 20.8 21.8 22 22 23 23.2 23.5 24.5 25.5 25.5 3 9 14.2 13 19.1 19.9 21 22 23 23.1 23.6 24.3 25.1 25.3 4 9.2 14.6 14 19.2 19.8 21 21 21 21.5 21.9 23 24.3 24.5 Average 9.3 14 14 19.8 20.4 22 22 22 22.5 23 23.5 24.8 24.7 PC 1 8.9 16 16 20.9 21.7 23 24 24 25.5 26.5 27.5 29.7 31.6 2 10.1 16.8 17 23.5 22.9 23 25 26 27.3 27.8 29.3 31.6 33.5 3 9.8 17.2 18 21.9 22.6 23 25 25 27.3 28.2 29.4 31.8 33.6 4 9 15.7 17 24.1 23.7 25 26 27 29.3 29.5 31.1 32.2 33.4 Average 9.5 16.4 17 22.6 22.7 24 25 26 27.4 28 29.3 31.3 33 SM 1 8.2 14.6 16 21.4 22.1 23 24 25 26.3 27.1 27.8 29.5 30.4 2 10 17.1 18 21.2 21.4 23 24 24 25.3 25.6 27.2 29.7 31.1 3 9.3 16.8 18 23.3 23.4 25 26 27 28 28.6 29.5 31.6 32.8 4 10.3 17.3 18 22.5 23.3 24 26 27 27.7 28 30.1 32.1 33.3 Average 9.5 16.5 17 22.1 22.6 24 25 26 26.8 27.3 28.7 30.7 31.9 high 1 9 15.3 16 19 19.6 21 22 22 22.8 23.8 23.8 24.6 25.1 2 9.2 16.1 17 21.8 21.2 23 25 25 24.2 25 26.5 28.6 29.5 3 9.4 16.4 17 20.3 19.6 21 22 22 24.1 24.7 25 27.2 27.7 4 9.1 16.2 18 20 19.5 21 22 23 23.2 23.7 23.1 25.1 25.3 Average 9.2 16 17 20.3 20 22 23 23 23.6 24.3 24.6 26.4 26.9 low 1 8.8 15.6 17 23.5 22 25 26 26 27.1 28.1 28.5 27 29.2 2 9.9 16.7 18 21.1 22.2 23 26 26 26.5 26.5 28.1 31.2 32.1 3 9.5 17.4 18 21.3 22 23 24 24 25.6 26.5 27.3 28.6 28.6 4 9.4 16.3 17 20 20.2 22 22 22 22.5 22.7 23.9 24.5 24.9 average 9.4 16.5 17 21.5 21.6 23 24 24 25.4 26 27 27.8 28.7
Lactobacillus rhamnosus Strain PL60 of the present invention has a body-fat reducing effect. Said Lactobacillus strain can be directly used as body-fat reducing functional foods for preventing or treating all diseases resulting from obesity, or can be used as additives of body-fat reducing functional foods.
Although the preferred embodiments of the present invention have been disclosed for illustrative purposes, those skilled in the art will appreciate that various modifications, additions and substitutions are possible, without departing from the scope and spirit of the invention as disclosed in the accompanying claims.
41844DNALactobacillus rhamnosus 1cgctggcggc gtgcctaata catgcaagtc gaacgagttc tgattattga aaggtgcttg 60catcttgatt taattttgaa cgagtggcgg acgggtgagt aacacgtggg taacctgccc 120ttaagtgggg gataacattt ggaaacagat gctaataccg cataaatcca agaaccgcat 180ggttcttggc tgaaagatgg cgtaagctat cgcttttgga tggacccgcg gcgtattagc 240tagttggtga ggtaacggct caccaaggca atgatacgta gccgaactga gaggttgatc 300ggccacattg ggactgagac acggcccaaa ctcctacggg aggcagcagt agggaatctt 360ccacaatgga cgcaagtctg atggagcaac gccgcgtgag tgaagaaggc tttcgggtcg 420taaaactctg ttgttggaga agaatggtcg gcagagtaac tgttgtcggc gtgacggtat 480ccaaccagaa agccacggct aactacgtgc cagcagccgc ggtaatacgt aggtggcaag 540cgttatccgg atttattggg cgtaaagcga gcgcaggcgg ttttttaagt ctgatgtgaa 600agccctcggc ttaaccgagg aagtgcatcg gaaactggra aacttgagtg cagaagagga 660cagtggaact ccatgtgtag cggtgaaatg cgtagatata tggaagaaca ccagtggcga 720aggcggctgt ctggtctgta actgacgctg aggctcgaaa gcatgggtag cgaacaggat 780tagataccct ggtagtccat gccgtaaacg atgaatgcta ggtgttggag ggtttccgcc 840cttc 844218DNAArtificial SequenceSynthetic primer used to identify Lactobacillus strains 2ctagcgggtg cgactttg 18321DNAArtificial SequenceSynthetic primer used to identify Lactobacillus strains 3ggccagctat gtattcactg a 21421DNAArtificial SequenceSynthetic primer used to identify Lactobacillus strains 4gcgatgcgaa tttctattat t 21
Patent applications by Tae Jin Kim, Seoul KR
Patent applications by Yeon-Hee Lee, Seoul KR
Patent applications in class Lactobacillus or Pediococcus or Leuconostoc
Patent applications in all subclasses Lactobacillus or Pediococcus or Leuconostoc